Immunomodulatory, insulinotropic, and cytotoxic activities of phylloseptins and

plasticin-TR from the Trinidanian leaf frog Phyllomedusa trinitatis

Jelena Pantic1 Laure Guilhaudis2 Vishal Musale3 Samir Attoub4 Miodrag L. Lukic1

Milena Mechkarska5 J. Michael Conlon3*

1Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences,

University of Kragujevac, Kragujevac, Serbia

2Normandy University, COBRA, UMR 6014 & FR 3038, Université de Rouen, INSA

Rouen, CNRS, 76821 Mont St Aignan, Cedex, France

3Diabetes Research Group, School of Biomedical Sciences, Ulster University, Coleraine

BT52 1SA, N. Ireland, U.K.

4Department of Pharmacology, College of Medicine and Health Sciences, United Arab

Emirates University, 17666 Al-Ain, United Arab Emirates

5Department of Life Sciences, The University of the West Indies, St Augustine, Trinidad

and Tobago

*Correspondence

J. Michael Conlon, Diabetes Research Group, School of Biomedical Sciences, Ulster

University, Cromore Rd, Coleraine BT52 1SA, N. Ireland, U.K.

E-mail: m.conlon@ulster.ac.uk Tel: 44-7918526277

1

ABSTRACT

The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and

insulin-releasing activities of seven phylloseptin-TR peptides and plasticin-TR, first

isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin-

1.1TR and phylloseptin-3.1TR showed greatest cytotoxic potency against A549, MDA-

MB231, and HT-29 human tumor-derived cells and against mouse erythrocytes.

Phylloseptin-4TR was the most hydrophobic and the most effective peptide at inhibiting

production of the pro-inflammatory cytokines TNF-α and IL-1β by mouse peritoneal cells

but was without effect on production of the anti-inflammatory cytokine IL-10.

Phylloseptin-2.1TR and phylloseptin-3.3TR were the most effective at stimulating the

production of IL-10. The non-cytotoxic peptide, plasticin-TR inhibited production of TNF-

α and IL-1β but was without effect on IL-10 production. The results of CD spectroscopy

suggest that the different properties of plasticin-TR compared with the immunostimulatory

activities of the previously characterized plasticin-L1 from Leptodactylus laticeps may

arise from greater ability of plasticin-TR to oligomerize and adopt a stable helical

conformation in a membrane-mimetic environment. All peptides stimulated release of

insulin from BRIN-BD11 rat clonal β-cells with phylloseptin-3.2TR being the most potent

and effective and phylloseptin-2.1TR the least effective suggesting that insulinotropic

potency correlates inversely with helicity. The study has provided insight into structure-

activity relationships among the phylloseptins. The combination of immunomodulatory and

insulinotropic activities together with low cytotoxicity suggests that phylloseptin 3.3-TR

and plasticin-TR may represent templates for the development of agents for use in anti-

inflammatory and Type 2 diabetes therapies.

2

Short Title: Immunomodulatory and insulinotropic phylloseptins and plasticin-TR

KEYWORDS

Amphibian skin peptide, Phylloseptin, Plasticin, Type 2 diabetes, Insulin-release, cytokine,

cytotoxicity

3

1 INTRODUCTION

Skin secretions of frogs belonging to the family Phyllomedusidae, currently comprising 65

species distributed in 8 genera1, are associated with a remarkably diverse array of

biologically active peptides.2-4 These include myotropic peptides such as bradykinins,

tachykinins, hyposins, and tryptophyllins as well as components that are structurally

similar, but not biosynthetically related, to neuroendocrine peptides found in mammals

such as cholecystokinin, corticotropin-releasing hormone, peptide tyrosine-tyrosine, and

opioid peptides. The precise physiological role of such peptides is unclear but it is

speculated that they act as a defense against ingestion by predators.5 In addition, a complex

arrays of peptides with varying degrees of cytotoxic activity against bacteria, fungi,

protozoa, and viruses are found in skin secretions of these frogs and examples include the

dermaseptins, dermatoxins, plasticins, phylloseptins, and phylloxins.2-4 Although originally

termed ‘antimicrobial peptides’ and considered to be a frontline defense against pathogenic

microorganisms in the environment, such peptides are multifunctional and also display

immunomodulatory properties6-8,, stimulate insulin release from clonal β-cells 9,10, and show

selective cytotoxicity against tumor cells11-13 and are therefore better described by the more

general term ‘host-defense peptides’.14

The Trinidadian leaf frog Phyllomedusa trinitatis Mertens, 1926 is widely

distributed in the coastal range mountains of Northern Venezuela up to approximately 1300

m above sea level and over much of the island of Trinidad.15 A recent study used reversed-

phase HPLC coupled with MALDI-TOF mass spectrometry and automated Edman

degradation to purify and characterize the extensive array of host-defense peptides present 4

in norepinephrine-stimulated secretions of this species.16 These comprised 15 dermaseptins,

9 phylloseptins, phyllocaerulein, a peptide with structural similarity to the plasticins, and a

putative antioxidant peptide. The study demonstrated that the phylloseptin-TR peptides

exhibited varying degrees of antimicrobial activity against Escherichia coli,

Staphylococcus epidermidis, and Candida albicans with phylloseptin-1.1TR, -2.1TR, and

3.1TR being the most potent. Plasticin-TR lacked antimicrobial activity at concentration up

to 100 µmol L-1.

Structure-activity relationships within the phylloseptin and plasticin families with

regard to immunomodulatory, insulinotropic and cytotoxic activities are incompletely

understood. Consequently, this study has investigated the biological activities of a series of

seven synthetic replicates of naturally occurring phylloseptin peptides and plasticin-TR that

were first isolated from skin secretions of P. trinitatis. The abilities of the peptides to

modulate production of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α)

and interleukin-1β (IL-1β) and the anti-inflammatory cytokine interleukin-10 (IL-10) by

mouse peritoneal cells, lyse human tumor-derived cells and mouse erythrocytes, and

stimulate the rate of insulin release in vitro from BRIN-BD11 rat clonal β-cells were

determined.

2 MATERIALS AND METHODS

2.1 Peptides

The phylloseptin and plasticin peptides used in this study were supplied in crude form by

EZBiolab Inc. (Carmel, IN, USA). The peptides were purified by reversed-phase HPLC on

a (2.2 cm x 25 cm) Vydac 218TP1022 (C-18) column equilibrated with acetonitrile/ 5

water/TFA (35.0/64.9.9/0.1, v/v/v) at a flow rate of 6 mL min-1. The concentration of

acetonitrile was raised to 63 % (v/v) over 60 min using a linear gradient. Absorbance was

measured at 214 nm and the major peak in the chromatogram was collected manually. The

identities of the peptides were confirmed by electrospray mass spectrometry and their final

purities were > 98%. The primary structures, molecular charges at pH 7, Grand Average of

Hydropathy (GRAVY) determined using the hydrophobicity scales of Kyte and Doolittle17,

and predicted helical domains of the peptides are shown in Table 1. Secondary structure

predictions were obtained using the AGADIR program which predicts the helical behavior

of monomeric peptides based on the helix/coil transition theory.18 Calculations were

performed at pH 7 and 278oK. A minimum percentage of 1% helicity/residue was

considered to predict the presence of a α-helix.

2.2 Effects on cytokine production

Experiments were performed on cells collected from the peritoneal cavities of unstimulated

8-week-old C57BL/6 mice under sterile conditions using 5 ml of cold phosphate-buffered

saline as previously described.19 Isolated cells, comprising a mixed population of immune

cells with predominance of macrophages and B cells, were suspended in Dulbecco's

Modified Eagle Medium culture medium containing 10% fetal bovine serum. Peptides (5

and 20 μg mL-1; approx. 2 and 8 nmol mL-1) were incubated with cells (2 x 105 cells/well)

for 24 h at 37°C in three independent incubations with 6 mice per group. After incubation,

cell-free supernatants were collected and kept at -20°C until time of analysis. Cytokine

concentrations were determined in triplicate using the following ELISA Duoset kits from R

& D Systems (Minneapolis, MN, USA) according to manufacturer’s recommended

6

protocols: tumor necrosis factor-alpha (TNF-α; DY410), interleukin-1β (IL-1β; DY401)

and interleukin-10 (IL-10; DY417).

2.3 Cytotoxicity studies

Human non-small cell lung adenocarcinoma A549 cells, human breast adenocarcinoma

MDA-MB-231 cells and human colorectal adenocarcinoma HT-29 cells were maintained

in culture as previously described.20 In all experiments, cell viability was higher than 99%

determined using trypan blue dye exclusion. Tumor cells were seeded in 96-well plates at

a density of 5 x 103 cells/well. After 24 h, all cells were treated for 24 h with increasing

concentrations of the peptides (0.1- 100 µmol L-1) in triplicate. The effect of the peptides

on cell viability was determined by measurement of ATP concentrations using a CellTiter-

Glo Luminescent Cell Viability assay kit (Promega Corporation, Madison, WI, USA).

Luminescent signals were measured using a GLOMAX Luminometer system. The LC50

value, calculated by non-linear regression analysis using commercially available software

(SPSS version 17.0; SPSS Inc., Chicago, IL, USA), was taken as the mean concentration of

peptide producing 50% cell death in three independent experiments.

All procedures involving animals were carried out in accordance with the UK

Animals (Scientific Procedures) Act 1986 and EU Directive 2010/63EU for animal

experiments and approved by Ulster University Animal Ethics Review Committee. In order

to determine hemolytic activity, peptides in the concentration range 7.8 - 500 μmol L-1

were incubated in triplicate with washed erythrocytes (2 x 107 cells) from NIH Swiss mice

in Dulbecco’s phosphate-buffered saline, pH 7.4 (100 µL) for 1 h at 37°C. After

centrifugation (12,000 x g for 15 s), the absorbance at 450 nm of the supernatant was

7

measured. A parallel incubation in the presence of 1% v/v Triton-X100 was carried out to

determine the absorbance associated with 100% hemolysis. The LC50 value was taken as

the mean concentration of peptide producing 50% hemolysis in three independent

experiments.

2.4 In vitro insulin release studies using BRIN-BD11 cells

BRIN-BD11 rat clonal β-cells21, maintained in culture as previously described 22, were

seeded into 24-well plates and allowed to attach during overnight incubation at 37 °C.

Incubations with purified synthetic peptides (10-12 to 10−6 mol L-1; n = 8) were carried out

for 20 min at 37°C in Krebs-Ringer bicarbonate (KRB) buffer supplemented with 5.6 mol

L-1 glucose as previously described.22 After incubation, aliquots of cell supernatant were

removed for insulin radioimmunoassay23. Control incubations were carried out in parallel

with the well-established insulin stimulatory agents 10 mmol L-1 alanine and 10 nmol L-1

glucagon-like peptide-1 (GLP-1). In order to investigate the effects of the peptides on the

integrity of the plasma membrane, peptides (10-7 - 3 x 10-6 mol L-1; n = 4) were incubated

with BRIN-BD11 cells for 20 min 37°C and the rate of lactate dehydrogenase (LDH)

release was measured using a CytoTox 96 non-radioactive cytotoxicity assay kit (Promega,

Southampton, UK) according to the manufacturer’s instructions as previously described.22

2.5 Circular dichroism spectroscopy

Spectra were recorded on a MOS-500 Circular Dichroism Spectrometer (Bio-Logic,

Seyssinet Pariset, France). Data points were collected from 260 to 185 nm, with an

8

integration time of 2 s per point and a step size of 1 nm, using a 1.0 mm path length

rectangular quartz cell. Measurements were carried out at room temperature. Plasticin-TR

was dissolved in 10 mmol L-1 sodium phosphate buffer, pH 7.0 and the solution used to

prepare samples containing 2,2,2-trifluoroethanol (TFE) (25%; 50%;75% v/v), methanol

(50%; 75%, 100% v/v), 20 mmol L-1 dodecylphosphocholine (DPC) and 20 mmol L-1

sodium dodecylsulfate (SDS). The concentration of 20 mmol L-1 for detergents was chosen

to ensure micelle formation. For the methanol-containing samples, the peptide was

dissolved in the pure solvent and diluted to the desired concentration. Peptide

concentrations of 0.2, 0.1 and 0.05 mg mL-1 were used for CD measurements. Three scans

were accumulated and averaged for each sample. All spectra were corrected by subtraction

of the background obtained for each peptide-free solution. Circular dichroism

measurements are reported as mean residue molar ellipticity ([θ]MRE (deg cm2 dmol−1).

Peptide -helical content was determined by using the Forood formula:

100×([]222/max[]222) with max[]222 =−40,000 [1−(2.5/n)], where n = number of amino

acid residues.24

2.6 Statistical Analysis

The distributions of data were evaluated for normality using Kolmogorov-Smirnov test and

then retested with Chi-Square test. Comparison of quantitative parametric data between

two study groups was done by application of unpaired t-test. Differences between the

paired data were evaluated using the paired t-test. A P-value of < 0.05, from two-tailed

tests, was considered statistically significant.

9

3 RESULTS

3.1 Effects of peptides on cytokine production

Production of the pro-inflammatory cytokine TNF-α by mouse peritoneal cells was

inhibited by incubation with all peptides with phylloseptins-2.1TR, -3.3TR, and-4TR and

plasticin-TR showing significant (P < 0.05) inhibition at 5 µg mL-1 (Fig.1A). Maximum

inhibition was achieved by phylloseptin-4TR and plasticin-TR. Incubation with these two

peptides also produced significant inhibition of the pro-inflammatory cytokine IL-1β at 5

µg mL-1 with phylloseptin-1.2TR and -3.3TR effective at 20 µg mL-1 (Fig. 1B). In contrast,

phylloseptin-4TR and plasticin-TR were without effect on the production of the anti-

inflammatory cytokine IL-10 whereas incubation with phylloseptin-1.2TR, -2.1TR,

-3.2TR, and -3.3TR significantly (P < 0.05) stimulated IL-10 production at a concentration

of 5 µg mL-1 (Fig. 1C) suggesting that these peptides are also anti-inflammatory.

3.2 Cytotoxicity studies

Cytotoxicity was initially determined by incubation of all peptides with human non-small

cell lung adenocarcinoma A549 cells and the results are shown in Table 2. Phylloseptin-

1.1TR, -2.1TR, and 3.1TR were the most potent (LC50 ≤ 20 µmol L-1) and so were

incubated with breast adenocarcinoma MDA-MB-231 cells and colorectal adenocarcinoma

HT-29 cells. The three peptides also showed relatively high cytotoxic potency (LC50 ≤ 38

µmol L-1) against these cell types. The concentration-dependence of the cytolytic activity

of phylloseptin-3.1TR is shown in Fig. 2. However, there was no selectivity towards

10

neoplastic cells as the three peptides were strongly cytotoxic against mouse erythrocytes

(LC50 ≤ 41 µmol L-1).

3.3 Effects of peptides on insulin-release from BRIN-BD11 cells

Control incubations of BRIN-BD11 cells with the well-established insulin secretagogues,

alanine (10 mmol L-1) and GLP-1 (10 nmol L-1) produced a 2- to 3-fold increase in the rate

of insulin release compared with the rate in the presence of glucose alone (Fig. 3). The

effects of incubation with the series of phylloseptin-TR peptides and with plasticin-TR are

shown in Fig. 3 and the data are summarized in Table 3. All peptides produced a

significant (P < 0.05) stimulatory response at concentrations ≥ 100 nmol L-1 with

phylloseptin-3.2TR being the most potent (threshold concentration 10 pmol L-1). This

peptide was also the most effective increasing the rate of insulin release by 2.3-fold at a

concentration of 3 µmol L-1. At concentrations up to and including 3 µmol L-1, no peptide

produced a significant increase in the rate of release of the cytosolic enzyme LDH from the

cells indicating that the integrity of the plasma membrane remained intact (data not

shown).

3.4 Conformational analysis of plasticin-TR

It has been reported that some plasticin peptides exhibit peptide-peptide association25 so

that it was necessary to determine the degree of solubility and aggregation of plasticin-TR

in each medium used. In the presence of organic solvent (TFE or MeOH) and at a

concentration of 0.1 mg mL-1, two types of behaviour were observed (Fig.S1): (A) an

increase in the overall ellipticity of the peptide with increasing % of MeOH showing a

11

stabilizing structural effect of the solvent and (B) a decrease in the overall ellipticity of the

peptide with increasing % of TFE percentage indicating limited solubility of plasticin-TR

in this solvent. Concentration effects on the range of 0.05 - 0.2 mg mL-1 (Figs S2 and S3)

were then evaluated in aqueous solution (10 mmol L-1 phosphate buffer at pH 7), in 100 %

MeOH, in 25% TFE, and in the presence of 20 mmol L-1 detergent (DPC or SDS).

Plasticin-TR showed a relatively small concentration dependence in aqueous solution, in

100% MeOH and in the presence of 20 mmol L-1 DPC. In 25% TFE and in the presence of

20 mmol L-1 SDS, the peptide exhibited concentration dependence of signal intensity

without modification of the overall aspect of the spectrum suggesting the formation of

oligomers. The increase of ellipticity with increase in concentration (0.05 - 0.1 mg mL-1 for

SDS; 0.1 - 0.2 mg/mL for 25% TFE) indicated that the auto-association of the peptide was

associated with a more stable structure. However, in the presence of SDS micelles, a

decrease of ellipticity was observed at a higher concentration (0.2 mg/mL) suggesting loss

of solubility of the resulting oligomers.

In order to compare the influence of the different environments on the conformation

of plasticin-TR, a 0.2 mg mL-1 concentration was chosen for all media except for SDS

micelles for which the 0.1 mg mL-1 concentration was used (Fig. 4). In aqueous solution,

the CD spectrum of the peptide displayed a single negative band centered at 197 nm,

indicative of random coil conformation. The CD spectra obtained in 25% TFE and in the

presence of detergents showed typical helical features with a positive peak around 192 nm

and double negative minima around 208 and 222 nm. In the presence of DPC, the -helical

contribution to the overall secondary structure of the peptide, estimated from the ellipticity

value at 222 nm, was 25%. This corresponds approximately to 5-6 residues out of 22 in

12

agreement with AGADIR predictions (Table 1). The α-helical content was similar in 25 %

TFE (26%) and appreciably higher in the presence of SDS (57 %). As the degree of

structuration in TFE and in the presence of SDS micelles was dependent on oligomer

formation (Fig. S3), it is difficult to make a direct comparison of the impact the

environment on the stabilization of the helical folding. The CD spectrum of plasticin-TR in

methanol was different from those obtained in the other media. The maximum positive

peak shifted toward a higher wavelength (196 nm) while the two negative minima shifted

toward a higher and a lower wavelength respectively (209 nm and 216 nm) indicating that

plasticin-TR adopted a mixture of helical and -sheet conformations. A helical content of

38% was deduced from the ellipticity at 222 nm. Although this value may be

overestimated, as the ellipticity value resulted from the presence of both -helical and -

sheet structures, it is clear that plasticin-TR is highly structured in methanol.

4 DISCUSSION

The phylloseptins are a family of structurally related, relatively small (17 - 20 amino acid

residues), cationic peptides that have a propensity to adopt an amphipathic α-helix in the

environment of a cell membrane or in a membrane-mimetic solvent.26 The phylloseptins

were first identified in skin secretions of the Brazilian tree-frogs Phyllomedusa

hypochondrialis (reclassified as Pithecopus hypochondrialis27) and Phyllomedusa oreades

(reclassified as Pithecopus oreades27) but subsequently have been shown to be produced by

numerous species belonging to the family Phyllomedusidae.28 The therapeutic potential of

13

the phylloseptins as anti-infective agents is suggested by their broad spectrum antibacterial

properties and the potent anti-plasmodial and anti-leishmanial activities of a phylloseptin

from Phyllomedusa azurea (reclassified as Pithecopus azureus1) indicate a possible role in

the treatment of malaria and leishmaniasis.29

The plasticins are glycine/leucine-rich peptides that contain multiple copies of the

GXXXG motif, where X is any amino acid, They derive their name from their

conformational flexible having the ability to adopt varying secondary structures depending

upon the environment.30 The plasticins were first identified in skin secretions of the

phyllomedusid frogs Agalychnis callidryas and Phyllomedusa biclor 31 but subsequently

plasticin-L1 was isolated from skin secretions of the South-American Santa Fe frog

Leptodactylus laticeps belonging to the family Leptodactylidae.32 Those plasticins with a

positive charge at physiological pH show broad-spectrum antimicrobial activity and

plasticin-L1 increased the production of the proinflammatory cytokines TNF-α, IL-1β, IL-

12 and IL-23 by mouse peritoneal macrophages but was without effect on the production of

anti-inflammatory IL-10.7

The present study has extended the scope of the phylloseptins and plasticins for

consideration as candidates for therapeutic intervention by demonstrating that all

phylloseptin-TR peptides and plasticin-TR inhibit production of TNF-α by mouse

peritoneal cells and phylloseptins-1.2 TR, -3.3TR, -4TR and plasticin-TR inhibit

production of IL-1β (Figs 1A and1B). The peptide producing the greatest inhibitory

response was phylloseptin-4TR. All peptides except phylloseptin-1.1TR and -4TR and

plasticin-TR stimulated production of the anti-inflammatory cytokine IL-10 with

phylloseptin-2.1TR and -3.3TR being the most effective. Previous studies have identified

14

other frog skin peptides, notably ascaphin-8, brevinin-2GU, B2RP-ERa, and rhinophrynin-

27, that inhibit TNF-α production either by mouse peritoneal cells or human peripheral

mononuclear cells.8 In contrast, frenatins-2D and 2.1S and esculentin-2CHa from frog skin

stimulate TNF-α production by these cell types.8 Similarly, B2RP-ERa, tigerinins-1M, -1R,

and -1V and hymenochirin-1B stimulate IL-10 production whereas alyteserin-2a, frenatin-

2.1S, and pseudhymenochirins-1Pb and -2Pa inhibit its production.8

It is not possible at this time to provide an unambiguous interpretation of these

findings in terms of their biological significance for the host. The ability of frogs to resist

infections by pathogenic microorganisms in the environment involves both innate and

adaptive immune defenses. Generation of effective immune responses depends on a

balance between pro- and anti-inflammatory responses so that an effective defense may be

determined by a balance between the effects of peptides that stimulate macrophages and

other cell types to produce both pro- and anti-inflammatory cytokines. Plasticin-TR and the

array of phylloseptins produced by P. trinitatis, by inhibiting TNF-α and IL-1β production

and stimulating IL-10 production, create a predominately immunosuppressive environment

that may serve to modulate an excessive and potentially harmful inflammatory response

triggered by exposure to pathogens. The immunomodulatory properties of the extensive

array of dermaseptins isolated from the P. trinitatis skin secretions remain to be

established.

In view of the marked difference in properties of the immunostimulatory plasticin-

L1 and the immunosupressive plasticin-TR, the secondary structures of the two peptides

were compared by CD spectroscopy. It has been shown that plasticin-L1, like other

15

members of the plasticin family,26,30 adopts a random coil conformation in water, a β-sheet

structure in methanol, and an α-helical conformation in 25% trifluoroethanol-water.32

The secondary structure of plasticin-TR is also solvent dependent but shows less

flexibility and variability than that of plasticin-L17,32 and other plasticins.25 Like plasticin-

L1, plasticin-TR adopts a random coil conformation in aqueous solution and an -helical

conformation in a 25% TFE-water mixture. Although, both peptides exhibit similar

features in the presence of TFE, the secondary structure content of the two peptides is

rather different. The mean molar residues ellipticities measured for plasticin-TR are higher

than the ones observed for plasticin-L1 ([]MRE at 222 nm is -9000 deg.cm2.dmol-1 for

plasticin-TR compared with -4500 deg.cm2.dmol-1 for plasticin-L1) demonstrating that

plasticin-TR exhibits a higher level of helical content. In contrast to plasticin-L1, plasticin-

TR adopts a conformation in methanol that is composed of a mixture of -helical and -

sheet structures. As observed for TFE, the mean molar residues ellipticities exhibited by

the peptide are higher in methanol than the ones measured for plasticin-L1 ([]MRE at 218

nm is -17000 deg.cm2.dmol-1 for plasticin-TR compared with -4500 deg.cm2.dmol-1 for

plasticin-L1) confirming a higher level of structured conformation. The CD spectra also

revealed that plasticin-TR adopts a more stable -helical conformation in the presence of

both anionic and zwitterionic membrane-mimetic micelles. The data provide evidence that

plasticin-TR exhibits a propensity to self-association and forms helical oligomers in

membrane-mimetic environments. Consequently, it is suggested, as one possibility, that the

very different cytokine-mediated immunomodulatory properties of plasticin-TR compared

with plasticin-L1 arise from its increased abilities to adopt a stable helical conformation

and to form oligomers in various media.

16

Several peptides isolated from phyllomedusid frogs have been considered as

candidates for development into anti-cancer agents. For example, dermaseptin L1 from the

lemur leaf frog Agalychnis lemur showed selective cytotoxicity to hepatocarcinoma HepG2

cells when compared with human erythrocytes12 and dermaseptin B2 and B3 from P.

bicolor (Phyllomedusinae) displayed both antitumor and angiostatic properties, inhibiting

proliferation of prostatic adenocarcinoma PC-3 cells as well as proliferation and

differentiation of bovine aortic endothelial cells.11 Phylloseptin-1.1TR, -2.1TR, and 3.1TR

represent the most cationic of the peptides studied (Table 1) and showed the greatest

cytotoxic potency against three human tumor-derived cell lines (Table 2). However, these

peptides showed little selectivity for neoplastic cells being strongly cytotoxic to

erythrocytes indicating that they possessed little therapeutic potential for development into

anti-cancer agents.

Plasticin-L1 and all phylloseptin peptides stimulated release of insulin from BRIN-

BD11 rat clonal β-cells and the study has shown that relatively small changes in the

primary structure of the phylloseptin molecule have marked effects upon insulinotropic

potency and effectiveness (Table 3). Phylloseptin-3.2 TR was the most potent peptide with

a threshold concentration of 10-11 mol L-1 and the most effective producing the greatest

increase in rate following incubation with a 3 µmol L-1. Phylloseptin-2.1TR was the least

potent (threshold concentration 3 µmol L-1) and least effective peptide.

Host-defense peptides from frog skin, with few exceptions, are cationic and adopt

an amphipathic α-helical conformation in a membrane-mimetic environment.4 The present

study has provided insight into structure-activity relationships among the phylloseptin

family. The factors that determine cytotoxicity of frog skin peptides against prokaryotic

17

and eukaryotic cells (cationicity, hydrophobicity, amphipathicity, and conformational

stability) are quite well understood 33 but the relative importance of parameters that

determine abilities to modulate cytokine production and stimulate insulin release are

largely unknown. Phylloseptin-4TR is the most effective peptide in attenuating production

of TNF-α and IL-β (Fig. 1) and, as shown in Table 1, this peptide is the most hydrophobic

of the phylloseptin peptides studied suggesting that this parameter may be of particular

importance in determining the abilities to inhibit production of proinflammatory cytokines.

In contrast, phylloseptin-4TR is largely ineffective in stimulating production of the anti-

inflammatory cytokine IL-10 whereas the more hydrophilic peptides phylloseptin-3.2TR

and 3.3TR produce a strong stimulation at concentrations as low as 5 µg mL-1 (Fig. 1). Use

of the AGADIR algorithm to predict helical content indicates that phylloseptin-2.1TR has

the propensity to form a stable α-helix whereas the predicted structure of phylloseptin-3.2

was a random coil. This suggests, but of course does not prove, that insulinotropic activity

of the phylloseptins varies inversely with the extent of peptide helicity.

Sepsis and septic shock carry a high morbidity and mortality burden, approximately

6 million fatalities worldwide per year, and are the major cause of death in intensive care

units.34 Production of both pro-inflammatory and anti-inflammatory cytokines by cells of

the innate immune system plays a critical role in regulation of immune responses during

sepsis and several immunosuppressive agents have shown limited therapeutic potential in

attenuating the early hyperinflammatory response that follows bacterial infection.35 The

importance of cationic host defense peptides in modulating the body's response to infection

and inflammation is increasingly being appreciated.36,37 In view of their the ability to

inhibit production of TNF-α and IL-1β and stimulate IL-10 production by peritoneal cells

18

coupled with very low toxicity against mouse erythrocytes and A549 human lung

adenocarcinoma cells, plasticin-TR and phylloseptin-3.3TR may represent templates for

the design of potent anti-inflammatory agents for use in the control hyperinflammatory

phase of sepsis. Elevated TNF-α induces insulin resistance in adipocytes and peripheral

tissues by impairing insulin signalling leading to development of Type 2 diabetes mellitus

(T2DM).38 Another critical event in the pathogenesis of T2DM is IL-1β-mediated

autoinflammation followed by β-cell death and subsequent loss of β-cell mass.

Neutralization of IL-1β or blockade of its receptor showed beneficial effects on the

restoring of β-cell function and even regeneration of islets.39 Consequently, blocking TNF-

α and IL-1β production by the plasticin and phylloseptin peptides in conjunction with their

ability to stimulate insulin release may represent one component of an effective strategy for

treatment of patients with insulin resistance and T2DM. In addition, suppression of

proinflammatory cytokine production, particular TNF-α and IL-1β, is an established

approach for the treatment of various autoimmune diseases.40 Given the low cytotoxicity of

plasticin-TR, further investigation of its therapeutic potential in this field may be

worthwhile.

A major disadvantage of peptide-based drugs, particularly if they are to be used for

systemic applications, is their rapid clearance from the circulation. Preliminary data

indicate that plasticin-TR and phylloseptin-3.3TR are rapidly degraded in mouse plasma

(unpublished observations). Esculentin-2CHa derivatives containing unnatural amino acids

[R7r,K15k,K23k]esculentin-2CHa(1-30) and incorporating fatty acids moieties [K15-

octanoate]esculentin-2CHa(1-30) are both more potent than the underivatized peptide in

vitro and more effective in vivo in terms of their glucose-lowering and insulin-releasing

19

activities.41.42 Future studies will focus on the development of long acting derivatives of

plasticin-TR and phylloseptin-3.3TR with regard to their therapeutic potential as anti-

inflammatory and anti-diabetic agents.

Acknowledgements

Funding for this study was provided by the Northern Ireland Department of Education and

Learning (DEL), Ulster University Strategic Funding, the University of the West Indies

Campus Research and Publication Fund (CRP.3.NOV16.8 grant #26600-457118), the

Ministry of Education, Science and Technological Development, Serbia (Grants ON

175069, ON 175071 and ON 175103) and Labex Synorg (ANR-11-LABX-0029).

20

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26

Legend to Figures

Fig. 1. Effects of incubation with phylloseptin-TR peptides and plasticin-TR (5 and 20 µg

mL-1) on the production of (A) TNF-α, (B) IL-1β, and (C) IL-10 by peritoneal cells from

C57BL/6 mice. Medium refers to incubation with medium only. Values show mean ±

SEM.,* P < 0.05.

Fig. 2. Effects of phylloseptin-3.1TR on the viability of (A) non-small cell lung

adenocarcinoma A549 cells, (B) breast adenocarcinoma MDA-MB-231 cells, and (C)

colorectal adenocarcinoma HT-29 cells after 24h exposure. All experiments were repeated

at least three times. Values show mean ± SEM.  

Fig. 3. Effects of increasing concentrations of (A) phylloseptin-1.2TR, (B) phylloseptin-

3.1TR, (C) phylloseptin-3.2TR, (D) phylloseptin-3.3TR, (E) phylloseptin-4TR, and (F)

plasticin-TR on the rate of release of insulin from BRIN-BD11 clonal β-cells compared

with 10 mmol L-1 alanine and 10 nmol L-1 GLP-1. Values show mean ± SEM with n = 8.

***p < 0.001, **p < 0.01, *p < 0.05 compared with glucose alone.

Fig. 4. Circular dichroism spectra of plasticin-TR at room temperature in aqueous solution

(solid black), 25 % TFE (dashed black), 100% MeOH (dotted black), and in the presence

of 20 mM DPC (dashed dotted gray) or 20 mM SDS (dashed gray).

27

Legends to supplementary figures

Fig. S1. CD spectra of plasticin-TR (0.1 mg mL-1) in 10 mmol L-1 sodium phosphate buffer,

pH 7.0 in the presence of increasing % of TFE (gray) and methanol (black)

Fig. S2. CD spectra of increasing concentrations of plasticin-TR in (A) 10 mol L-1 sodium

phosphate buffer, pH 7, (B) 20 mmol L-1 DPC in 10 mmol L-1 sodium phosphate buffer, pH

7 and (C)100% methanol. For clarity, spectra at 0.05 mg mL-1 in methanol and 20 mmol L-1

DPC are not shown.

Figure S3: CD spectra of increasing concentrations of plasticin-TR in 10 mmol L-1 sodium

phosphate buffer pH 7 in the presence of (A) 25% TFE and (B) 20 mmol L-1 SDS.

28

Table 1. Primary structure and physicochemical properties of the phylloseptins and

plasticin-TR used in this study

Net charge GRAVY Helical at pH 7 domain

PS-1.1TR FLSLIPKIAGGIASLVKNLa +3 1.26 12-18

PS-1.2TR FLSLIPKIAGGIASLVKDLa +2 1.26 Non-helical

PS-2.1TR FLSLIPHIATGIAALAKHLa +2.2 1.31 10-19

PS-3.1TR FFSMIPKIATGIASLVKNLa +3 1.10 12-18

PS-3.2TR FFSMIPKIATGIASLVKDLa +2 1.10 Non-helical

PS-3.3TR FFSMIPKIATGIASLVKNL +2 1.10 Non-helical

PS-4TR LLGMIPVAITAISALSKLa +2 1.78 11-18

Plasticin-TR GLVSGLLNSVTGLLGNLAGGGL 0 1.12 10-15

GRAVY represents Grand Average of Hydropathy determined using the hydophobicity

scales of Kyte and Doolittle.17 Secondary structure predictions were obtained using the

AGADIR program which predicts the helical behavior of monomeric peptides.18

C-terminal α-amidation is denoted by a.

29

Table 2. Cytotoxicities of phylloseptin-TR peptides and plasticin-TR against lung

adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, colorectal

adenocarcinoma HT-29 cells, and human red blood cells (RBC).

Data show mean LC50 values (μmol L-1). ND not determined

30

Peptide A549 MDA-MB231 HT-29 RBC

PS-1.1TR 20 20 34 28

PS-1.2TR 55 ND ND 75

PS-2.1TR 20 24 38 41

PS-3.1TR 18 18 27 12

PS-3.2TR 65 ND ND 110

PS-3.3TR >100 ND ND 340

PS-4TR >100 ND ND 65

Plasticin-TR >100 ND ND >500

Table 3. Effects of phylloseptins and plasticin-TR on the rate of insulin release from

BRIN-BD11 clonal β-cells

Peptide Threshold

Conc. (mol L-1)Max. effect

ng/106 cells/20min

None NA 0.35 ± 0.01

Phylloseptin-1.1TR 10-7 0.61 ± 0.06**

Phylloseptin-1.2TR 10-9 0.62 ± 0.05**

Phylloseptin-2.1TR 3 x10-6 0.43 ± 0.05*

Phylloseptin-3.1TR 10-9 0.65 ± 0.04**

Phylloseptin-3.2TR 10-11 0.80 ± 0.07**

Phylloseptin-3.3TR 10-10 0.69 ± 0.06**

Phylloseptin-4TR 10-10 0.73 ± 0.06**

Plasticin-TR 10-10 0.63 ± 0.05**

Threshold concentration refers to the minimum concentration of peptide producing a

significant increase in the rate of insulin release compared with the rate in the presence of

glucose only. Max. effect refers to the rate of insulin release in the presence of 3 µmol L-1

peptide. NA: not applicable, **P<0.001, *P<0.05 compared to 5.6 mmol L-1 glucose alone.

31

Fig.1

32

Fig. 2

33

Fig. 3

34

Fig. 4

35

Fig. S1

36

Fig. S2

37

Fig. S3

Graphical Abstract

38